PROJECT SUMMARY: The dietary benefits of tryptophan have been classically linked to its property as an essential amino acid required by mammals for protein synthesis, but recent work in the past decade has revealed its equally essential role as an important source of metabolites for the microbial community to generate signals, mainly via the Aryl Hydrocarbon Receptor (AHR), to affect a plethora of cellular functions. AHR signaling pathway is highly promiscuous not only responding to diverse ligands with resembling structures, but also crosstalk with and is modulated by numerous cellular signaling pathways. Distinct agonists can result in ligand selective differences in AHR structure and function, ultimately leading to agonist-selective gene expression and biological effects. In the gut, AHR activation by bacterial tryptophan catabolites influences mucosal homeostasis and microbiota composition. Although bacterial tryptophan metabolites and AHR axis may contribute to microbial sensing and tolerance, it is unclear if probiotics and pathobionts produce distinct tryptophan metabolites thereby eliciting bacterial species- and agonist-specific host mucosal modulating effects. Both Lactobacillus and Ruminoccocus spp catabolize tryptophan to a variety of tryptamine and indolic products that bind at low to high affinities to the AHR. The identities and specific biological effects of tryptophan metabolites produced by the probiotic Lactobacillus rhamnosus GG (LGG) and the pathobiont R. gnavus are currently unknown. Preliminary RNA-Seq analysis of mouse intestinal mucosa perfused with live LGG revealed a robust induction of AHR signaling activity as well as anti-inflammatory cytokines. Bacterial 16s rRNA sequencing and untargeted fecal metabolomics screening of a newly engineered Lyz1 knockout mouse model revealed a dysbiosis featured by expansion of mucolytic bacterial species (e.g., R. gnavus), accompanied by a pronounced elevation of tryptophan metabolites and a changed epithelial cell composition. The hypothesis is that the probiotic LGG and the pathobiont R. gnavus metabolize dietary tryptophan into distinct agonists that elicit differential mucosal modulating effects via the host AHR signaling. Aim I will use mono-colonization of germ-free mice, dietary tryptophan depletion, unbiased metabolomics profiling, and isogenic mutant bacteria deficient in tryptophan-metabolizing to identify LGG and R. gnavus specific tryptophan metabolites and their differential mucosal modulating effects. Aim 2 will employ organoid culture, intraluminal perfusion of AHR agonists, and genetic ablation of host AHR signaling to determine the direct effects of bacterial species-specific tryptophan metabolites and AHR signaling on Lyz1-deficient mouse mucosal homeostasis and colitis susceptibility. This MPI project uses rigorous approaches, including gnotobiotics, bacterial genetics, dietary manipulation, and unbiased metabolomics, etc. to address an important mechanism involving diet-microbe-host interaction that may underlie the biological natures of probiotics and pathobionts. The two PIs have a history of productive collaboration, and have demonstrated experiences in studying intestinal physiology. This project employing innovative animal models is significant for public health and disease intervention.